Microelectronic device

ABSTRACT

A method of detecting a nucleic acid (e.g., DNA, RNA) that contains at least one preselected base (e.g., adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine) comprises (a) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction; (b) detecting the oxidation-reduction reaction; and (c) determining the presence or absence of the nucleic acid from the detected oxidation-reduction reaction at the preselected base. The method may be used in a variety of applications, including DNA sequencing, diagnostic assays, and quantitative analysis.

This application is a divisional of U.S. Pat. application Ser. No.08/667,338, filed Jun. 20, 1998 now issued as U.S. Pat. No. 5,871,918and is a continuation-in-part of application Ser. No. 08/495,817 filedJun. 27, 1995, now abandoned, and is a continuation-in-part of copendingapplication Ser. No. 60/016,265 filed Apr. 19, 1996, and 60/060,949filed Jun. 27, 1995 the disclosures of which are incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to nucleic acid hybridization andsequencing, and particularly to methods of qualitatively andquantitatively detecting nucleic acid hybridization and to methods ofnucleic acid sequencing.

BACKGROUND OF THE INVENTION

The detection of individual DNA sequences in heterogenous samples of DNAprovides a basis for identifying genes, DNA profiling, and novelapproaches to DNA sequencing. One approach to DNA hybridizationdetection involves the use of surface bound DNA sequences which can beassayed using an analytical response that indicates hybridization of thesurface-bound oligomer to a sequence in the heterogeneous sample. Theseanalytical methods generally involve laser-induced fluorescence arisingfrom a covalently attached label on the target DNA strand, which is notsensitive to single-base mismatches in the surface-bound duplex. Forexample, U.S. Pat. Nos. 5,143,854 and 5,405,783 to Pirrung et al.;Fodor, et al., Nature 364:555 (1993); Bains, Angew. Chem. 107:356(1995); and Noble, Analytical Chemistry 67(5):201A (1995) proposesurfaces or "chips" for this application. In an alternate method,proposed by Hall, et al., Biochem. and Molec. Bio. Inter. 32(1):21(1994), DNA hybridization is detected by an electrochemical methodincluding observing the redox behavior of a single stranded DNA ascompared to a double stranded DNA. This technique is also not sensitiveto single-base mismatches in the DNA sample. Techniques for detectingsingle-base mismatches include enzymatic or chemical cleavage studies,such as those proposed in U.S. Pat. No. 5,194,372 to Nagai et al.However, these techniques are disadvantageous inasmuch as they requiremore time and separation technology.

U.S. Pat. No. 5,312,527 to Mikkelson et al. describes a voltammetricsequence-selective sensor for detecting target nucleic acid in which adouble-stranded nucleic acid is contacted to a redox-active complex. Thecomplex binds non-specifically to the double-stranded DNA. Because thecomplex itself is the redox-active compound that provides a voltammetricsignal, the complex does not function in a catalytic manner without theaddition of an enzyme.

U.S. Pat. No. 4,840,893 to Hill et al. describes an electrochemicalassay for nucleic acids in which a competitive binding event between aligand and an antiligand is in turn detected electrochemically.

Accordingly, there remains a need in the art for a method of detectingDNA hybridization, including a method of detecting single-base pairmismatches, which is both rapid and sensitive, and which can be rapidlyapplied on-line.

SUMMARY OF THE INVENTION

In general, the present invention provides a method of detecting anucleic acid that contains at least one preselected base (e.g., adenine,guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine). Themethod comprises (a) reacting the nucleic acid with a transition metalcomplex capable of oxidizing the preselected base in anoxidation-reduction reaction; (b) detecting the oxidation-reductionreaction; and (c) determining the presence or absence of the nucleicacid from the detected oxidation-reduction reaction at the preselectedbase. Depending on the particular embodiment of the method and theparticular object desired, the method may optionally include the step ofcontacting the nucleic acid with a complementary nucleic acid to form ahybridized nucleic acid.

As a first aspect, the present invention provides a method of detectingDNA hybridization. The method includes (a) contacting a DNA sample withan oligonucleotide probe to form a hybridized DNA, (b) reacting thehybridized DNA with a transition metal complex capable of oxidizing apreselected base in the oligonucleotide probe in an oxidation-reductionreaction where the oligonucleotide probe has at least one of thepreselected bases, (c) detecting the oxidation-reduction reaction, (d)determining the presence or absence of hybridized DNA from the detectedoxidation-reduction reaction at the preselected base. As discussed indetail below, the step of detecting the oxidiation-reduction reactionmay, in general, be carried out by measuring electron flow from thepreselected base.

As a second aspect, the present invention provides another method ofdetecting DNA hybridization. The method includes (a) contacting a DNAsample with an oligonucleotide probe to form a hybridized DNA, (b)reacting the hybridized DNA with a transition metal complex capable ofoxidizing a preselected base in the oligonucleotide probe in anoxidation-reduction reaction, where the oligonucleotide probe has atleast one of the preselected bases, (c) detecting theoxidation-reduction reaction, (d) measuring the reaction rate of thedetected oxidation-reduction reaction, (e) comparing the measuredreaction rate to the oxidation-reduction reaction rate of the transitionmetal complex with a single-stranded DNA, and then (f) determiningwhether the measured reaction rate is essentially the same as theoxidation-reduction reaction rate of the transition metal complex withsingle-stranded DNA.

As a third aspect, the present invention provides an apparatus fordetecting DNA hybridization. The apparatus includes (a) a plurality ofDNA sample containers, (b) sample handling means for carrying theplurality of DNA sample containers, (c) an oligonucleotide probedelivery means for delivering the oligonucleotide probe to each of theDNA sample containers, (d) a transition metal complex delivery means fordelivering the transition metal complex to each of the plurality of DNAsample containers, and (e) an oxidation-reduction reaction detector fordetecting an oxidation-reduction reaction.

As a fourth aspect, the present invention provides a second apparatusfor detecting DNA hybridization. The apparatus includes (a) a DNA samplecontainer, (b) an oligonucleotide probe delivery means for delivering aplurality of oligonucleotide probes to the DNA sample container, (c) atransition metal complex delivery means for delivering the transitionmetal complex to the DNA sample container, and (d) anoxidation-reduction reaction detector for detecting anoxidation-reduction reaction.

As a fifth aspect, the present invention provides a method of sequencingDNA. The method includes (a) contacting a DNA sample with anoligonucleotide probe to form a hybridized DNA, where theoligonucleotide probe includes a preselected synthetic base having aunique oxidation potential, (b) reacting the hybridized DNA with atransition metal complex capable of oxidizing the preselected syntheticbase in the oligonucleotide probe in an oxidation-reduction reaction,where the oligonucleotide probe has a predetermined number of thepreselected synthetic bases, (c) detecting the oxidation-reductionreaction, (d) measuring the reaction rate of the detectedoxidation-reduction reaction, and (e) identifying the base paired withthe preselected synthetic base.

The foregoing and other aspects of the present invention are explainedin detail in the detailed description set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cyclic voltammograms of Ru(bpy)₃ ²⁺ with and withoutcalf thymus DNA. The solid line represents the scan of 50 μM Ru(bpy)₃ ²⁺at 25 mV/s in 700 mM NaCl/50 mM sodium phosphate buffer. The dotted linerepresents the voltammogram of 50 μM Ru(bpy)₃ ²⁺ and 3.0 mM (nucleotide)calf thymus DNA.

FIG. 2 shows the cyclic voltammograms of Ru(bpy)₃ ²⁺ in the presence of5'-AAATATAGTATAAAA as a single strand (C) and hybridized tocomplementary strands (A & B). The scan rate is 25 mV/s. (A) represents25 μM Ru(bpy)₃ ²⁺ 100 μM (in guanine nucleotides) double stranded fullyhybridized DNA (5'-AAATATAGTATAAAA)•(3'-TTTATATCATATTTT). (B) representsRu(bpy)₃ ²⁺ with a duplex containing a GA mismatch(5'-AAATATAGTATAAAA)•(3'-TTTATATAATATTTT), and (C) represents Ru(bpy)₃²⁺ a single strand containing one guanine nucleotide(5'-AAATATAGTATAAAA).

FIG. 3 is a schematic illustration of one illustrative apparatus usefulfor carrying out the methods of the present invention.

FIG. 4 is a schematic illustration of a detection method particularlyadvantageous for the quantitative detection of DNA where the preselectedbase is located on the target nucleic acid.

FIG. 5 shows the cyclic voltammograms of Ru(bpy)₃ ²⁺ (25 μM) at a scanrate of 25 mV/s in 50 mM sodium phosphate buffer with 0.7M NaCl, pH 7.(A) No added oligonucleotide. (B) With 75 μM d[5'-TTTTATACTATATTT]. (C)With 75 μM of the hybrid of the oligomer from B andd[5'-GGGAAATATAGTATAAAAGGG]. Working electrode: tin-doped indium oxide.Reference electrode: Ag/AgCl. Counter electrode: Pt wire. The secondarystructure of the hybrid from C is indicated on the Figure.

FIG. 6 shows the cyclic voltammograms of (A) Ru(bpy)₃ ²⁺ (25 μM), (B)Ru(bpy)₃ ²⁺ (25 μM) with inosine 5'-monophosphate (0.3mM), and (C)Ru(bpy)₃ ²⁺ (25 μM) with guanosine 5'-monophosphate. Structures ofinosine and guanine are shown in the Figure.

FIG. 7 schematically illustrates an alternate embodiment of theinvention of FIG. 4, where the preselected bases are on an elongationproduct of terminal transferase.

FIG. 8 schematically illustrates an alternate embodiment of theinvention of FIG. 4, carried out in a sandwich assay format.

FIG. 9 is a schematic illustration by top plan view of a microelectronicdevice useful for carrying out methods of the present invention.

FIG. 10 is a side sectional view of a portion of the device illustratedin FIG. 9.

FIG. 11 shows the cyclic voltammograms using nylon-modified ITOelectrodes, of Ru(bpy)₃ ²⁺ (200 μM) at buffer-soaked nylon, Ru(bpy)₃ ²⁺(200 μM) at DNA-soaked nylon in high salt (700 mM added NaCl) buffer,and Ru(bpy)₃ ²⁺ (200 μM) at DNA-soaked nylon in low salt (i.e., no addedNaCl) buffer.

FIG. 12 shows the cyclic voltammograms of Os(bpy)₃ ²⁺ (200 μM) usingnylon-modified ITO electrodes soaked in buffer or in DNA. FIG. 12A showsthe cyclic voltammogram with 700 mM NaCl added. FIG. 12B shows thecyclic voltammogram with no NaCl added.

FIG. 13 shows the cyclic voltammograms at nylon-modified ITO electrodesshowing cyclic voltammograms of Ru(bpy)₃ ²⁺ (200 μM) at buffer-soakednylon, Ru(bpy)₃ ²⁺ (200 μM) at tRNA-soaked nylon in high salt (700 mMadded NaCl) buffer, and Ru(bpy)₃ ²⁺ (200 μM) at tRNA-soaked nylon in lowsalt (no added NaCl) buffer.

FIG. 14 shows the cyclic voltammogram of Ru(bpy)₃ ²⁺ (25 μM) alone andwith (100 μM in strands) of 5'-AAATATAG_(n) TATAAAA where n=1 (G), 2(GG), or 3 (GGG). The scan rate is 25 mV/s.

FIG. 15 shows the cyclic voltammogram of Ru(bpy)₃ ²⁺ (25 μM) alone andwith (100 μM in strands) of 5'-AAATAT(AGT)_(n) ATAAAA where n=1, 2, or3. The scan rate is 25 mV/s.

FIG. 16 shows the cyclic voltammogram of 25 μMRuthenium(4,4'-dimethylbipyridine)₃ ²⁺ (or "Ru(4,4'-Me₂ -bpy)₃ ²⁺ ")alone (solid) and with (100 μM in strands) of 5'-AAATATAGTATAAAA(dotted) and 5'-AAATATAGGGTATAAAA (dashed). The scan rate is 25 mV/s.

FIG. 17 shows the cyclic voltammogram of 0.20 mM Ru(4,4'-Me₂ -bpy)₃ ²⁺in 50 mM sodium phosphate buffer (ph 7) with 0.7M NaCl at a scan rate of25 mV/s. Curve (A) represents Ru(4,4'-Me₂ -bpy)₃ ²⁺ alone. Curve (B)represents Ru(4,4'-Me₂ -bpy)₃ ²⁺ in the presence of 0.70 mM6-mercaptoguanosine 5'-monophosphate.

FIG. 18 shows cyclic voltammograms of 200 μM of Ru(bpy)₃ ²⁺ at ITOworking electrodes to which a Hybond N+ nylon membrane is attached.Membranes are impregnated with poly[C] and subjected to thehybridization protocol in buffer (A) and a concentrated solution ofpoly[G] (B).

FIG. 19 shows cyclic voltammograms of 200 μM of Ru(bpy)₃ ²⁺ at ITOworking electrodes to which a Hybond N+ nylon membrane is attached.Membranes are impregnated with poly[C] and subjected to thehybridization protocol in buffer (A) and a concentrated solution ofdenatured calf thymus DNA (B).

FIG. 20 shows the cyclic voltammograms (scan rate=25 mV/s) of 200 μMRu(bpy)₃ ²⁺ at a nylon-modified glassy carbon electrode (A) without DNAor (B) after adsorption of DNA to the nylon film.

DETAILED DESCRIPTION OF THE INVENTION

The term "nucleic acid" as used herein refers to any nucleic acid,including both DNA and RNA. Nucleic acids of the present invention aretypically polynucleic acids; that is, polymers of individual nucleotidesthat are covalently joined by 3', 5' phosphodiester bonds.

The term "complementary nucleic acid" as used herein refers to anynucleic acid, including oligonucleotide probes, that specifically bindsto another nucleic acid to form a hybridized nucleic acid.

The phrase "determining the presence or absence of" is intended toinclude both qualitatively determining and quantitatively determiningthe presence or absence of the detected event (e.g., DNA hybridization,RNA hybridization, detecting target nucleic acid, etc.).

The terms "hybridized DNA" and "hybridized nucleic acid" refer to asingle-stranded DNA which is hybridized to form a double-stranded DNA ornucleic acid, or a double-stranded DNA or nucleic acid which ishybridized to form triple helix DNA or nucleic acid.

While the methods and apparatus of the present invention are sometimesexplained with respect to DNA herein, this is for purposes of clarity,and it is to be understood that the methods and apparatus of the instantinvention may be applied to other nucleic acids such as RNA.

A. Nucleic Acid Amplification Methods

Inasmuch as the processes of the present invention involve contactingthe DNA sample to an oligonucleotide probe to produce a hybridized DNA,it may be desirable for certain applications to amplify the DNA prior tocontacting with the probe. Amplification of a selected, or target,nucleic acid sequence may be carried out by any suitable means. Seegenerally D. Kwoh and T. Kwoh, Am. Biotechnol. Lab. 8, 14-25 (1990).Examples of suitable amplification techniques include, but are notlimited to, polymerase chain reaction (including, for RNA amplification,reverse-transcriptase polymerase chain reaction), ligase chain reaction,strand displacement amplification, transcription-based amplification(see D. Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173-1177 (1989)),self-sustained sequence replication (or "3SR") (see J. Guatelli et al.,Proc. Natl. Acad. Sci. USA 87, 1874-1878 (1990)), the Qβ replicasesystem (see P. Lizardi et al., Biotechnology 6, 1197-1202 (1988)),nucleic acid sequence-based amplification (or "NASBA") (see R. Lewis,Genetic Engineering News 12 (9), 1 (1992)), the repair chain reaction(or "RCR") (see R. Lewis, supra), and boomerang DNA amplification (or"BDA") (see R. Lewis, supra). The bases incorporated into theamplification product may be natural or modified bases (modified beforeor after amplification), and the bases may be selected to optimizesubsequent electrochemical detection steps.

Polymerase chain reaction (PCR) may also be carried out in accordancewith known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; and 4,965,188 (the disclosure of all U.S. Patent referencescited herein are to be incorporated herein by reference). In general,PCR involves, first, treating a nucleic acid sample (e.g., in thepresence of a heat stable DNA polymerase) with one oligonucleotideprimer for each strand of the specific sequence to be detected underhybridizing conditions so that an extension product of each primer issynthesized which is complementary to each nucleic acid strand, with theprimers sufficiently complementary to each strand of the specificsequence to hybridize therewith so that the extension productsynthesized from each primer, when it is separated from its complement,can serve as a template for synthesis of the extension product of theother primer, and then treating the sample under denaturing conditionsto separate the primer extension products from their templates if thesequence or sequences to be detected are present. These steps arecyclically repeated until the desired degree of amplification isobtained. Detection of the amplified sequence may be carried out byadding to the reaction product an oligonucleotide probe capable ofhybridizing to the reaction product (e.g., an oligonucleotide probe ofthe present invention), the probe carrying a detectable label, and thendetecting the label in accordance with known techniques. Where thenucleic acid to be amplified is RNA, amplification may be carried out byinitial conversion to DNA by reverse transcriptase in accordance withknown techniques.

Strand displacement amplification (SDA) may be carried out in accordancewith known techniques. See generally G. Walker et al., Proc. Natl. Acad.Sci. USA 89, 392-396 (1992); G. Walker et al., Nucleic Acids Res. 20,1691-1696 (1992). For example, SDA may be carried out with a singleamplification primer or a pair of amplification primers, withexponential amplification being achieved with the latter. In general,SDA amplification primers comprise, in the 5' to 3' direction, aflanking sequence (the DNA sequence of which is noncritical), arestriction site for the restriction enzyme employed in the reaction,and an oligonucleotide sequence (e.g., an oligonucleotide probe of thepresent invention) which hybridizes to the target sequence to beamplified and/or detected. The flanking sequence, which serves tofacilitate binding of the restriction enzyme to the recognition site andprovides a DNA polymerase priming site after the restriction site hasbeen nicked, is preferably about 15 to 20 nucleotides in length; therestriction site is functional in the SDA reaction (i.e.,phosphorothioate linkages incorporated into the primer strand do notinhibit subsequent nicking--a condition which may be satisfied throughuse of a nonpalindromic recognition site); the oligonucleotide probeportion is preferably about 13 to 15 nucleotides in length.

Ligase chain reaction (LCR) is also carried out in accordance with knowntechniques. See, e.g., R. Weiss, Science 254, 1292 (1991). In general,the reaction is carried out with two pairs of oligonucleotide probes:one pair binds to one strand of the sequence to be detected; the otherpair binds to the other strand of the sequence to be detected. Each pairtogether completely overlaps the strand to which it corresponds. Thereaction is carried out by, first, denaturing (e.g., separating) thestrands of the sequence to be detected, then reacting the strands withthe two pairs of oligonucleotide probes in the presence of a heat stableligase so that each pair of oligonucleotide probes is ligated together,then separating the reaction product, and then cyclically repeating theprocess until the sequence has been amplified to the desired degree.Detection may then be carried out in like manner as described above withrespect to PCR.

B. Oligonucleotide Probes

As noted above, the processes of the present invention are useful fordetecting the hybridization of DNA. The first step of the processinvolves contacting a DNA sample with an oligonucleotide probe to form ahybridized DNA. The oligonucleotide probes which are useful in themethods of the present invention can be, any probe comprised of betweenabout 4 or 6 bases up to about 80 or 100 bases or more, more preferablybetween about 8 and about 15 bases. Oligonucleotide probes may beprepared having any of a wide variety of base sequences according totechniques which are well known in the art. Suitable bases for preparingthe oligonucleotide probe may be selected from naturally occurringnucleotide bases such as adenine, cytosine, guanine, uracil, andthymine; and non-naturally occurring or "synthetic" nucleotide basessuch as 7-deazroquanine,8-oxo-guanine, 6-mercaptoguanine,4-acetylcytidine, 5-(carboxyhydroxyethyl)uridine, 2'-O-methylcytidine,5-carboxymethylamino-methyl-2-thioridine,5-carboxymethylaminometlyl)uridine, dihydrouridine,2'-O-methylpseudouridine, β,D-galactosylqueosine, 2'-O-methylguanosine,inosine, N6-isopentenyladenosine, 1-methyladenosine,1-methylpseeudouridine, 1-methylguanosine, 1-methylinosine,2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine,3-methylcytidine, 5-methylcytidine, N6-methyladenosine,7-methylguanosine, 5-methylaminomethyluridine,5-methoxyaminomethyl-2-thiouridine, β,D-mannosylqueosine,5-methoxycarbonylmethyluridine, 5-methoxyuridine,2-methylthio-N6-isopentenyladenosine,N-((9-β-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine,N-((9-β-D-ribofuranosylpurine-6-yl)N-methyl-carbamoyl)threonine,uridine-5-oxyacetic acid methylester, uridine-5-oxyacetic acid,wybutoxosine, pseudouridine, queosine, 2-thiocytidine,5-methyl-2-thiouridine, 2-thiouridine, 2-thiouridine, 5-methylurdine,N-((9-β-D-ribofuranosylpurine-6-yl)carbamoyl)threonine,2'-O-methyl-5-methyluridine,2'-O-methyluridine, wybutosine, and3-(3-amino-3-carboxypropyl)uridine. Any oligonucleotide backbone may beemployed, including DNA, RNA (although RNA is less preferred than DNA),modified sugars such as carbocycles, and sugars containing 2'substitutions such as fluoro and methoxy. The oligonucleotides may beoligonucleotides wherein at least one, or all, of the internucleotidebridging phosphate residues are modified phosphates, such as methylphosphonates, methyl phosphonothioates, phosphoromorpholidates,phosphoropiperazidates and phosphoramidates (for example, every otherone of the internucleotide bridging phosphate residues may be modifiedas described). The oligonucleotide may be a "peptide nucleic acid" suchas described in P. Nielsen et al., Science 254, 1497-1500 (1991). Theonly requirement is that the oligonucleotide probe should possess asequence at least a portion of which is capable of binding to a knownportion of the sequence of the DNA sample. It may be desirable in someapplications to contact the DNA sample with a number of oligonucleotideprobes having different base sequences (e.g., where there are two ormore target nucleic acids in the sample, or where a single targetnucleic acid is hybridized to two or more probes in a "sandwich" assay).

C. Hybridization Methodology

The DNA (or nucleic acid) sample may be contacted with theoligonucleotide probe in any suitable manner known to those skilled inthe art. For example, the DNA sample may be solubilized in solution, andcontacted with the oligonucleotide probe by solubilizing theoligonucleotide probe in solution with the DNA sample under conditionswhich permit hybridization. Suitable conditions are well known to thoseskilled in the art (See, e.g., U.S. Pat. No. 4,358,535 to Falkow et al.and other U.S. Patent references citing the same) and include high saltconcentration conditions. Alternatively, the DNA sample may besolubilized in solution with the oligonucleotide probe immobilized on asolid support, whereby the DNA sample may be contacted with theoligonucleotide probe by immersing the solid support having theoligonucleotide probe immobilized thereon in the solution containing theDNA sample.

D. Oxidizing Agents and Oxidation-Reduction Reactions

When a hybridization step precedes the oxidizing step, then afterhybridization the hybridized DNA (or nucleic acid) is then reacted witha suitable oxidizing agent which is capable of oxidizing a preselectedbase in the oligonucleotide probe in an oxidation-reduction reaction.The preselected base can be any naturally occurring or syntheticnucleotide base in the oligonucleotide probe which undergoes oxidationupon reaction with the selected oxidizing agent. The preselected baseexhibits a unique oxidation rate when paired as compared to when thepreselected base is unpaired. The preselected base should exhibit uniqueoxidation rates when paired with each of the four naturally occurringbases. Generally, bases whose 5'-mononucleotides (e.g., the5'-deoxyribonucleotide or 5'-ribonucleotide) exhibit rate constantsabove 10⁴ M⁻¹ s⁻¹ can be detected using the catalytic reaction. Examplesof suitable preselected bases include but are not limited to guanine,adenine, 8-oxo-guanine, and 8-oxo-adenine, 8-bromo-guanine, guanosine,xanthosine, wyosine, pseudouridine, 6-mercaptoguanine,8-mercaptoguanine, 2-thioxanthine, 6-thioxanthine, 6-mercaptopurine,2-amino-6-carboxymethyl-mercaptopurine, 2-mercaptopurine,6-methoxypurine, 2-acetylamino-6-hydroxypurine,6-methylthio-2-hydroxypurine, 2-dimethylamino-6-hydroxypurine,2-hydroxypurine, 2-aminopurine, 6-amino-2-dimethylallyl-purine,2-thioadenine, 8-bydroxyadenine, 8-methoxyadenine. Typically, thepreselected base is selected from the group consisting of guanine,adenine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine, withguanine being the currently preferred naturally occurring preselectedbase and 6-mercaptoguanine the currently preferred synthetic preselectedbase.

The oxidizing agent may be any charged molecule such as a cationic,anionic, or zwitterionic molecule which is reactive with the preselectedbase at a unique oxidation potential. Thus the selection of oxidizingagent will be dependent upon the particular preselected base chosen, andwill be readily determinable by those skilled in the art. Particularlypreferred oxidizing agents include transition metal complexes which arecapable of metal-DNA electron transfer with the preselected base suchthat the reduced form of the metal complex is regenerated, completing acatalytic cycle. Examples of suitable transition metal complexes for usein the methods of the present invention include, for example,Ruthenium²⁺ (2,2'-bipyridine)₃ ("Ru(bpy)₃ ²⁺ "), Ruthenium²⁺(4,4'-dimethyl-2,2'-bipyridine)₃ ("Ru(Me₂ -bpy)₃ ²⁺ "), Ruthenium²⁺(5,6-dimethyl-1,10-phenanthroline)₃ ("Ru(Me₂ -phen)₃ ²⁺ "), Iron²⁺(2,2'-bipyridine)₃ ("Fe(bpy)₃ ²⁺ "), Iron²⁺ (5-chlorophenanthroline)₃("Fe(5-Cl-phen)₃ ²⁺ "), Osmium²⁺ (5-chlorophenanthroline)₃("Os(5-Cl-phen)₃ ²⁺ "), dioxorhenium¹⁺ phosphine, and dioxorhenium¹⁺pyridine ("ReO₂ (py)₄ ¹⁺ "). Some anionic complexes useful as oxidizingagents are: Ru(bpy)((SO₃)₂ -bpy)₂ ²⁻ and Ru(bpy)((CO₂)₂ bpy)₂ ²⁻ andsome zwitterionic complexes useful as oxidizing agents are Ru(bpy)₂((SO₃)₂ -bpy) and Ru(bpy)₂ ((CO₂)₂ -bpy) where (SO₃)₂ -bpy²⁻ is4,4'-disulfonato-2,2',bipyridine and (CO₂)₂ -bpy²⁻ is4,4'-dicarboxy-2,2'-bipyridine. Suitable substituted derivatives of thepyridine, bypyridine and phenanthroline groups may also be employed incomplexes with any of the foregoing metals. Suitable substitutedderivatives include but are not limited to 4-aminopyridine,4-dimethylpyridine, 4-acetylpyridine, 4-nitropyridine,4,4'-diamino-2,2'-bipyridine, 5,5'-diamino-2,2'-bipyridine,6,6'-diamino-2,2'-bipyridine, 4,4'-diethylenediamine-2,2'-bipyridine,5,5'-diethylenediamine-2,2'-bipyridine,6,6'-diethylenediamine-2,2'-bipyridine, 4,4'-dihydroxyl-2,2'-bipyridine,5,5'-dihydroxyl-2,2'-bipyridine, 6,6'-diihydroxyl-2,2'-bipyridine,4,4',4"-triamino-2,2',2"-terpyridine,4,4',4"-triethylenediamine-2,2',2"-terpyridine,4,4',4"-trihydroxy-2,2',2"-terpyridine,4,4',4"-trinitro-2,2',2"-terpyridine,4,4',4"-triphenyl-2,2',2"-terpyridine, 4,7-diamino-1,10-phenanthroline,3,8-diamino-1,10-phenanthroline,4,7-diethylenediamine-1,10-phenanthroline,3,8-diethylenediamine-1,10-phenanthroline,4,7-dihydroxyl-1,10-phenanthroline, 3,8-dihydroxyl-1,10-phenanthroline,4,7-dinitro-1,10-phenanthroline, 3,8-dinitro-1,10-phenanthroline,4,7-diphenyl-1,10-phenanthroline, 3,8-diphenyl-1,10-phenanthroline,4,7-disperamine-1,10-phenanthroline,3,8-disperamine-1,10-phenanthroline, anddipyrido[3,2-a:2',2'-c]phenazine.

The oxidizing agent may be reacted with the hybridized DNA according toany suitable technique, to effect the oxidation-reduction reaction ofthe oxidizing agent with the preselected base. All that is required isthat the oxidizing agent be reacted with the hybridized DNA sample underconditions sufficient to effect the selective oxidation of thepreselected base. For example, the transition metal may be reacted withsolubilized hybridized DNA by solubilizing the oxidizing agent in thesolution containing the solubilized hybridized DNA under conditionssufficient to permit the oxidation-reduction reaction between theoxidizing agent and the preselected base to occur. Alternatively, in theembodiment wherein the hybridized DNA is immobilized on a solid support,the oxidizing agent may be reacted with the hybridized DNA byimmobilizing the oxidizing agent on the same solid support and immersingthe solid support in a solution under conditions sufficient to permitthe oxidation-reduction reaction of the oxidizing agent and thepreselected base to occur. The solvent in which the oxidation-reductionreaction takes place may be any suitable solvent for solubilizing DNA,and preferably comprises water. Suitable conditions for permitting theoxidation-reduction reaction to occur will be known to those skilled inthe art.

In a hybridized DNA or nucleic acid, the oxidizing agents dock in theminor groove of DNA, thus intimate contact between the preselected baseand the oxidizing agent is precluded by the unique structure of thedouble (or triple) helix. This protection of the preselected baseresidue results in the necessity of electron tunneling through solvent,which attenuates the rate of electron transfer. The solventaccessibility varies with the nature of the nucleotide base which ispaired with the preselected base. The tunneling distance can beestimated according to the formula:

    k/k.sub.ss =exp(-βΔr)

where Δr is the change in distance in the duplex compared to the singlestrand and k_(ss) is the rate constant for oxidation of the preselectedbase in the single-stranded DNA sample. Thus, the tunneling distancebetween the preselected base and the oxidizing agent is different foreach base pairing and for unpaired DNA. Therefore, the electron-transferrate constant indicates the identity of the paired (or mismatched) base.If the driving force for electron transfer is significantly less thanthe reorganizational energy (λ), a plot of RT In k against drivingforce, corrected for work terms associated with the approach of thereactants, yields a straight line with a slope of 1/2, according toMarcus theory. Based on Marcus theory then, the absolute rate constantscan be calculated by the following equation:

    k=v exp[-β(r-r.sub.0)]exp[-(ΔG+λ).sup.2 /4λRT]

wherein v is the rate constant in the diffusion-controlled limit (10¹¹M⁻¹ s⁻¹), r is the distance between the reactant and product in theactivated complex, r₀ is the distance of closest approach of reactantand product, and β is the influence of the intervening medium. Because,as noted above, the preselected base is incorporated into the interiorof the hybridized DNA, this imposes a finite distance across which theelectron must tunnel to the oxidizing agent. Thus, r does not equal r₀.β for water is about 3 Å⁻¹. This relatively large value for β indicatesthat significant changes in the electron-transfer rate constants will beeffected by very small changes in the tunneling distance. Since the DNAconformation between the preselected base and the base paired with thepreselected base is dependent upon the base paired with the preselectedbase, the base paired with the preselected base affects the tunnelingdistance through which the electron must tunnel between the preselectedbase and the oxidizing agent. A correlation between the tunnelingdistance and the specific base paired with the preselected base istherefore established.

E. Detection of Oxidation-Reduction Reactions

The occurrence of the oxidation-reduction reaction may be detectedaccording to any suitable means known to those skilled in the art. Forexample, the occurrence of the oxidation-reduction reaction may bedetected using a detection electrode to observe a change in theelectronic signal which is indicative of the occurrence of theoxidation-reduction reaction. Typically, a detection electrode which issensitive to the transfer of electrons between the oxidizing agent andthe hybridized DNA is placed in contact with the solution containing thereacted hybridized DNA and oxidizing agent. Generally, a referenceelectrode and an auxiliary electrode are also placed in contact with thesolution in conjunction with the detection electrode (with most of thecurrent passing through the auxiliary electrode). Suitable detectionelectrodes will be well known to those skilled in the art and include,for example, a glassy carbon electrode or an indium tin oxide electrode.Similarly, suitable reference electrodes will also be known in the artand include, for example, silver/silver chloride electrodes.

The detection of the electronic signal associated with theoxidation-reduction reaction permits the determination of the presenceor absence of hybridized DNA. The step of determining the presence orabsence of hybridized DNA typically includes (i) measuring the reactionrate of the oxidation-reduction reaction, (ii) comparing the measuredreaction rate to the oxidation-reduction reaction rate of the transitionmetal complex with a single-stranded DNA, and then (iii) determiningwhether the measured reaction rate is essentially the same as theoxidation-reduction reaction rate of the transition metal complex withsingle-stranded DNA. The step of measuring the reaction rate may becarried out by any suitable means. For example, the relative reactionrate may be determined by comparing the current as a function of scanrate, probe concentration, target concentration, mediator, buffer,temperature, and/or electrochemical method.

The oxidation-reduction reaction rate may be measured according tosuitable means known to those skilled in the art. Typically, theoxidation-reduction reaction rate is measured by measuring theelectronic signal associated with the occurrence of theoxidation-reduction reaction. For example, the electronic signalassociated with the oxidation-reduction reaction may be measured byproviding a suitable apparatus in electronic communication with thedetection electrode. A suitable apparatus will be capable of measuringthe electronic signal which is generated so as to provide a measurementof the oxidation-reduction reaction rate of the reaction of thehybridized DNA and the oxidizing agent. The electronic signal may becharacteristic of any electrochemical method, including cyclicvoltammetry, normal pulse voltammetry, chronoamperometry, andsquare-wave voltammetry, with cyclic voltammetry being the currentlypreferred form.

The measured reaction rate may then be compared to the knownoxidation-reduction reaction rate of the transition metal complex with asingle-stranded DNA. As discussed in detail above, the tunnelingdistance between the oxidizing agent and the selected base in either thehybridized or single-stranded DNA affects the oxidation-reductionreaction rate of the reaction between the oxidizing agent and thepreselected base. Accordingly, hybridized DNA exhibits a differentoxidation-reduction reaction rate than single-stranded DNA. The presenceor absence of hybridized DNA at the preselected base can be determinedby determining whether or not the measured oxidation-reduction reactionrate is the same as the oxidation-reduction reaction rate of theoxidizing agent and the preselected base in single-stranded DNA.Furthermore, the tunneling distance between the oxidizing agent and thepreselected base will differ according to the bond distance between thepreselected base and its pair, such that each possible base pairing maybe distinguished from the others. The bond distance between thepreselected base and its base pair is dependent upon the base which ispaired with the preselected base. For example, the oxidation-reductionreaction rate for the oxidation of guanine paired with adenine differsfrom the oxidation-reduction reaction rate for the oxidation of guaninepaired with cytosine, which in turn is different front theoxidation-reduction reaction rate for the oxidation of guanine pairedwith guanine, which is also different from the oxidation-reductionreaction rate for the oxidation of guanine paired with thymine. Morespecifically, the oxidation-reduction reaction rates for the oxidationof guanine follow the trend wherein single strand guanine is greaterthan guanine paired with adenine, which is greater than guanine pairedwith guanine, which is greater than guanine paired with thymine, whichis greater than guanine paired with cytosine. Accordingly, the methodsof the present invention are useful for detecting single-base pairmismatches at the preselected base or at the base pair adjacent to thepreselected base.

Advantageously, the distinction between the oxidation-reduction reactionrates of the oxidation of the preselected base when paired with each ofthe various naturally occurring bases also permits the identification ofthe base paired with the preselected base. The base paired with thepreselected base may be identified by (i) measuring the reaction rate ofthe detected oxidation-reduction reaction, (ii) comparing the measuredreaction rate to each of the four different known oxidation-reductionreaction rates of the oxidizing agent with a DNA having adenine,cytosine, guanine, or thymine bound to the preselected base, and (iii)determining which of the known oxidation-reduction reaction rates isessentially the same as the measured reaction rate. The reaction ratemay be measured according to techniques described above. Similarly, thereaction rates of each of the four different oxidation-reductionreactions of the oxidizing agent with a DNA having adenine, cytosine,guanine or thymine bound to the preselected base may be measuredaccording to the same techniques such that these reaction rates areknown. The measured reaction rate of the oxidation-reduction reaction ofthe oxidizing agent with the hybridized DNA may then be compared to theknown oxidation-reduction reaction rates of the oxidizing agent with aDNA having adenine, cytosine, guanine or thymine bound to thepreselected base. For example, the base paired with the preselected baseis determined by determining the known base pairing having theoxidation-reduction reaction rate which is essentially the same as themeasured oxidation-reduction reaction rate.

F. DNA Sequencing

The present invention also provides a method of sequencing DNAcomprising (a) contacting a DNA sample with an oligonucleotide probe toform a hybridized DNA, the oligonucleotide probe including a preselectedsynthetic base having a unique oxidation potential; (b) reacting thehybridized DNA with an oxidizing agent such as a transition metalcomplex, capable of oxidizing the preselected synthetic base in theoligonucleotide probe in an oxidation-reduction reaction, theoligonucleotide probe having a predetermined number of the preselectedsynthetic bases; (c) detecting the oxidation-reduction reaction; (d)measuring the reaction rate of the detected oxidation-reductionreaction; and (e) identifying the base paired with the preselectedsynthetic base.

As in the methods discussed hereinabove, the DNA sample may be amplifiedprior to the step of contacting with the oligonucleotide probe,according to techniques known to those skilled in the art. The syntheticbase may be selected from the group of bases described hereinabove, andother synthetic bases known to those skilled in the art. The onlylimitation is that the synthetic base should possess a unique oxidationpotential as compared with the oxidation potentials of the fournaturally occurring bases, i.e., adenine, cytosine, guanine, andthymine. The steps of contacting the DNA sample with the oligonucleotideprobe; reacting the hybridized DNA with the oxidizing agent, detectingthe oxidation-reduction reaction, and measuring the reaction rate may becarried out as described hereinabove. The step of identifying the basepaired with the preselected synthetic base includes the steps of (i)comparing the measured reaction rate to each of the four different knownoxidation-reduction reaction rates of the oxidizing agent with the DNAhaving adenine, cytosine, guanine, or thymine bound to the preselectedsynthetic base; and (ii) determining which of the knownoxidation-reduction reaction rates is essentially the same as themeasured reaction rate.

In another embodiment, the oligonucleotide probe further includes asecond preselected synthetic base. The second preselected synthetic basehas a unique oxidation potential which is different from the oxidationpotential of the first preselected synthetic base. In this embodiment,the step of detecting the oxidation-reduction reaction of the oxidizingagent with the preselected base further includes detecting theoxidation-reduction reaction of the oxidizing agent with the secondpreselected synthetic base as well. In addition, the step of measuringthe oxidation-reduction reaction rate further includes measuring theoxidation-reduction reaction rate of the oxidation of the secondpreselected base by the oxidizing agent as well. Further, the step ofidentifying the base paired with the preselected synthetic base furtherincludes identifying the base paired with the second preselectedsynthetic base as well. According to this embodiment, theoxidation-reduction reactions of both preselected bases may be detectedsuch that ultimately the bases which are paired with each preselectedsynthetic base may be identified using the method described hereinabove.As will be apparent to those skilled in the art, the foregoing methodmay be carried out with more than two preselected synthetic bases,provided that each preselected synthetic base exhibits a uniqueoxidation potential which is different from the oxidation potential ofall other preselected synthetic bases, and different from the oxidationpotential of each of the four naturally occurring bases.

Inasmuch as each base which is paired with a preselected base may beidentified according to the methods described herein, DNA may besequenced by repeating the steps of the foregoing method with asufficient number of different oligonucleotide probes having thepreselected synthetic base at different sites to identify each base inthe DNA sample. In other words, the DNA sample may be sequenced byproviding a sufficient number of oligonucleotide probes wherein eachprobe sequence includes at least one of the preselected synthetic bases,and the synthetic base is located at a different and calculated sitealong the probe sequence in each oligonucleotide probe. In this manner,repeated detection of the oxidation-reduction reaction of the hybridizedDNA with an oxidizing agent, measurement of the oxidation-reductionreaction rate, and identification of the base paired with thepreselected synthetic base will result in a base-by-base identificationof the sequence of the DNA sample.

G. Apparatus

The present invention also provides apparatus useful for carrying outthe methods of the present invention. One such illustrative apparatus isschematically illustrated in FIG. 3. In general, the apparatus comprisesa plurality of DNA sample containers 10. A drive assembly 11 serves as asample handling means for carrying the plurality of DNA samplecontainers. A liquid reservoir 12, a feed line 13 and a valve 14 serveas an oligonucleotide probe delivery means for delivering theoligonucleotide probe to each of the DNA sample containers, and acorresponding liquid reservoir 15, feed line 16 and valve 17 serves asan oxidizing agent delivery means for delivering the transition metalcomplex to each of the plurality of DNA sample containers. A probeassembly 20 including a drive 21 and a probe 22 serves as anoxidation-reduction reaction detector means for detecting anoxidation-reduction reaction. In operation, DNA samples arepre-deposited in the sample containers 10. The drive assembly 11 thentransports consecutively the sample containers 10 beneath theoligonucleotide probe delivery means and the oxidizing agent deliverymeans for delivering the respective reagents therein. After reagentdelivery, the respective sample container is advanced by the drive meansto a position beneath the probe 22 and the probe 22 advanced by thedrive 21 into the sample container for detection of theoxidation-reduction reaction. Additional electrodes necessary forcarrying out of the cyclic voltammogram are carried with the probe 22.Operation of the various components and collection of data may becarried out with a suitable controller 30, such as a software programrunning on a general purpose computer.

Numerous variations on the foregoing apparatus will, of course, bereadily apparent to those skilled in the art. The plurality of DNAsample containers may be any suitable container known to those skilledin the art, and includes microtiter plates, test tubes, petri dishes,culture vials, solid supports, and the like, which are capable ofcontaining the DNA sample. The sample handling means may be any suitablydesigned sample container handling means known to those skilled in theart, which is capable of carrying the DNA sample containers.

Suitable oligonucleotide probe delivery means for delivering theoligonucleotide probe to each of the DNA sample containers are wellknown in the art. For example, according to one embodiment, theoligonucleotide probe delivery means comprises a solid support on whichthe oligonucleotide probe is immobilized. The oligonucleotide probedelivery means should permit sufficient contact between the DNA sampleand the oligonucleotide probe under appropriate conditions to effecthybridization of the DNA sample and the oligonucleotide probe. Suitableoxidizing agent delivery means for delivering the oxidizing agent toeach of the plurality of DNA sample containers are well known in theart. For example, according to one embodiment, the oxidizing agent isattached to a solid support which comprises the oxidizing agent deliverymeans. The oxidation-reduction reaction detector for detecting anoxidation-reduction reaction may, according to one embodiment, compriseone or more electrodes which are capable of detecting the oxidation ofthe preselected base. Suitable detection electrodes and referenceelectrodes are described hereinabove with reference to the methods ofthe present invention. Preferably, the electrodes are in electroniccommunication with a means for measuring the oxidation-reductionreaction rate of the oxidation-reduction reaction. Suitable means formeasuring the oxidation-reduction reaction rate are known to thoseskilled in the art as described hereinabove.

In an alternate embodiment of the apparatus of the present invention,the apparatus for detecting DNA hybridization comprises (a) a DNA samplecontainer; (b) an oligonucleotide probe delivery means for delivering aplurality of oligonucleotide probes to the DNA sample container; (c) anoxidizing agent delivery means for delivering the oxidizing agent to theDNA sample container; and (d) an oxidation-reduction reaction detectorfor detecting an oxidation-reduction reaction. This apparatus is adaptedfor use with immobilized probes such as those described in U.S. Pat.Nos. 5,143,854 and 5,405,783 to Pirrung et al.; Fodor, et al., Nature364:555 (1993); Bains, Agnew. Chem. 107:356 (1995); and Noble,Analytical Chemistry 67(5):21 (1995), the disclosures of which areincorporated herein by reference in their entirety.

As noted above, the DNA sample container may be any suitable containerknown to those skilled in the art. The oligonucleotide probe deliverymeans is preferably a solid support having a plurality ofoligonucleotide probes immobilized thereon, which is capable ofdelivering the probes to the DNA sample container. For example,according to one embodiment, the solid support having the plurality ofoligonucleotide probes immobilized thereon is contacted with the DNAsample within the DNA sample container under conditions sufficient topermit the hybridization of the DNA sample with one or moreoligonucleotide probes.

Suitable oxidizing agent delivery means for delivering the oxidizingagent to the DNA sample container are described hereinabove. Thepreferred oxidizing agent delivery means comprises a solid supporthaving the oxidizing agent immobilized thereon. According to onepreferred embodiment, the oxidizing agent and the plurality ofoligonucleotide probes are immobilized on the same solid support.

The apparatus according to the present invention are useful forperforming diagnostic assays of a variety of DNA samples. The pluralityof oligonucleotide probes permits the assay and detection of a varietyDNA within a single sample, thus providing a useful tool for thescreening of a single sample for a variety of DNA including pathogens,viruses, and the like.

H. RNA Hybridization Detection, RNA Sequencing, and RNA MismatchDetection

Also disclosed herein are methods of detecting RNA hybridization, RNAsequencing methods, and methods of detecting RNA mismatches. RNA usefulfor carrying out such methods, includes, but is not limited to,ribosomal RNA, transfer RNA, or genomic RNA (e.g., RNA obtained from RNAviruses such as retroviruses, HIV-1, etc.). A first aspect of theinstant invention is, accordingly, a method of detecting RNAhybridization comprises: (a) contacting an RNA sample with anoligonucleotide probe to form a hybridized RNA; (b) reacting thehybridized RNA with a transition metal complex capable of oxidizing apreselected base in the oligonucleotide probe in an oxidation-reductionreaction, the oligonucleotide probe having at least one of thepreselected bases; (c) detecting the oxidation-reduction reaction; (d)determining the presence or absence of hybridized RNA from the detectedoxidation-reduction reaction at the preselected base.

More particularly, a method of detecting RNA hybridization comprises:(a) contacting an RNA sample with an oligonucleotide probe to form ahybridized RNA; (b) reacting the hybridized RNA with a transition metalcomplex capable of oxidizing a preselected base in the oligonucleotideprobe in an oxidation-reduction reaction, the oligonucleotide probehaving at least one of the preselected bases; (c) detecting theoxidation-reduction reaction; (d) measuring the reaction rate of thedetected oxidation-reduction reaction; (e) comparing the measuredreaction rate to the oxidation-reduction reaction rate of the transitionmetal complex with a single-stranded RNA; and then (f) determiningwhether the measured reaction rate is the same as theoxidation-reduction reaction rate of the transition metal complex withsingle-stranded RNA.

A method of sequencing RNA comprises: (a) contacting an RNA sample withan oligonucleotide probe to form a hybridized RNA, the oligonucleotideprobe including a preselected base having a unique oxidation rate; (b)reacting the hybridized RNA with a transition metal complex capable ofoxidizing the preselected base in the oligonucleotide probe in anoxidation-reduction reaction, the oligonucleotide probe having apredetermined number of the preselected bases; (c) detecting theoxidation-reduction reaction; (d) measuring the reaction rate of thedetected oxidation-reduction reaction; and (e) identifying the basepaired with the preselected base.

Oligonucleotide probes, hybridization methodology, oxidizing agents,detection of oxidation reduction reactions, and apparatus useful forcarrying out these methods are essentially as given in sections A-Habove, adapted for use with RNA as the nucleic acid sample in accordancewith principles known to those skilled in the art (e.g., uracil replacesthymine as a base).

I. Detection of Preselected Base on Target Nucleic Acid

In the methods specifically described above, metal complexes are used toobtain an electrochemical current from single- and double-stranded DNAor nucleic acids. Preselected bases such as guanine give anelectrochemical signal, and this signal is much weaker fordouble-stranded DNA. Such methods advantageously exhibit high structuralsensitivity, and can resolve a single base mismatch. Such methods aretherefore particularly advantageous for the sequencing of DNA. However,two drawbacks of such methods are that: (a) there is a negative signalon going from the probe strand to the hybrid, and (b) there is noamplification of the signal. The following techniques provide solutionsto these problems. In addition, the following techniques areparticularly useful for diagnostic assays, and are particularly usefulfor the the quantitative detection of nucleic acids.

In view of the foregoing, also disclosed herein is a method of detectingthe presence or absence of a target nucleic acid in a test samplesuspected of containing the same, wherein the target nucleic acidcontains at least one preselected base. In contrast to the methodsdescribed above, in the instant method the preselected base is locatedon the target nucleic acid, rather than on the oligonucleotide probe.

The method may be carried out on a test sample containing the targetnucleic acid. Any test sample suspected of containing the target nucleicacid may be used, including (but not limited to) tissue samples such asbiopsy samples and biological fluids such as blood, sputum, urine, andsemen samples, bacterial cultures, soil samples, food samples, etc. Thetarget nucleic acid may be of any origin, including animal, plant ormicrobiological (e.g., viral, prokaryotic and eukaryotic bacterial,protozoal, fungal, protoctistal, etc.) depending on the particularpurpose of the test. The sample may be processed or purified prior tocarrying out the instant method in accordance with techniques known orapparent to those skilled in the art; and nucleic acids therein may bedigested, fragmented, and/or amplified (see above) prior to carrying outthe instant method, if so desired.

As schematically illustrated in FIG. 4, the method comprises (a)contacting the felt sample to an oligonucleotide probe that specificallybinds to the target nucleic acid to form a hybridized nucleic acid; (b)contacting the hybridized nucleic acid to a transition metal complexthat oxidizes the preselected base in an oxidation-reduction reaction;(c) detecting the presence or absence of the oxidation-reductionreaction associated with the hybridized nucleic acid; and (d)determining the presence or absence of the target nucleic acid in thetest sample from the detected oxidation-reduction reaction at thepreselected base. As illustrated in FIG. 4, the oligonucleotide probemay be immobilized on a solid support to facilitate separating the testsample from the hybridized nucleic acid, with the separating stepoccurring prior to the detecting step (e.g., between steps (a) and (b)or between steps (b) and (c)). Alternatively, the oligonucleotide probemay be provided free in solution, and other means provided to separatethe hybridized nucleic acid from the sample (e.g., by a mediator nucleicacid that binds to the oligonucleotide probe, or by a biotin-avidinbinding interaction, where biotin is bound to the oligonucleotide probeand avidin is immobilized on a solid support).

Preferably, the target nucleic acid contains at least ten more of thepreselected base than does the oligonucleotide probe, or more preferablyat least 50 or 100 more of the preselected base than does theoligonucleotide probe. A larger current enhancement is advantageouslyobtained when the target nucleic acid contains many more of thepreselected base than does the oligonucleotide probe.

Optionally, but preferably, the oligonucleotide probe is free of thepreselected base, or is at least essentially free of the preselectedbase (i.e., contains sufficiently less of the preselected base thatsignal from probe does not interfere with or is not mistaken as a signalfrom the target nucleic acid). Where a sequence of naturally occuringbases is not available that will conveniently hybridize to the targetnucleic acid, the strategy of employing alternate bases that are redoxinactive (discussed below) may be employed.

The target nucleic acid is preferably longer than the oligonucleotideprobe, and at least one of the preselected base is not hybridized to theoligonucleotide probe in the hybridized nucleic acid (i.e., is an"overhanging" base), as illustrated in FIG. 4. Preferably, at least 10,50, or 100 of the preselected bases are "overhanging" bases, therebyproviding substantial amplification of the electrochemical signaldetected.

For example, an oligonucleotide probe that does not contain any guanineresidues (e.g., only A, T, and C) may be used. The cyclic voltammogramof Ru (bpy)₃ ²⁺ in the presence of this strand is very similar to thatwithout the oligomer. This strand is then hybridized to a target strandthat contains guanines in either, or both (as illustrated in FIG. 4 by a"G" adjacent the target nucleic acid strand), the overlappingbase-paired regions or in overhanging regions if the target nucleic acidis longer than the oligonucleotide probe. Because multiple guanines aredetected, the signal is amplified relative to the number of hybridsformed. In a case where a genomic DNA or RNA is the target strand, largenumbers of overhanging guanines are encountered, which would givetremendous signal amplification. For example, ribosomal RNA may containas many as 1,000 guanines for a particular organism, and would thereforeprovide approximately a 1,000-fold amplification per hybridizationevent.

For example, in one preferred embodiment, the assay for the preselectedbase on the target strand involves immobilization of the (preferablyredox-silent) probe strand on a solid surface oriented close to theelectrode surface, which provides a low background signal when scannedin the presence of the mediator. The solid surface is then contactedwith a solution of the target strand, which contains the preselectedbase. If hybridization occurs, the target strand will now be in closeproximity to the electrode, and a current enhancement will be detected.

Quantitating nucleic acids. The instant method is particularly wellsuited to the quantitative detection of nucleic acids. In the casesdescribed in this section, the rate constant for oxidation of the hybridby the oxidizing agent (e.g., Ru(bpy)₃ ³⁺) can be determined from thecyclic voltammogram (or other electronic signal) by digital simulation.Under most conditions this reaction will obey second-order kinetics, sorate k=[Ru(bpy)₃ ²⁺ ][DNA] where k is the rate constant that is specificfor the particular probe-target hybrid, [Ru(bpy)₃ ²⁺ ] is theconcentration of the oxidizing agent, and [DNA] is the concentration ofthe hybrid (which could be a DNA-RNA hybrid). If k and [Ru(bpy)₃ ²⁺ ]are known, then the quantity of the hybrid can be determined. Inpractice, a calibration curve for current enhancements obtained withdifferent quantities of standard solutions containing target DNA or RNAis constructed and the current enhancement used to obtain the quantityof hybrid directly. This quantity is then related directly to thequantity of target material (e.g., infectious organism in a clinicalsample). See, e.g., M. Holodniy et al., J. Virol. 69, 3510-3516 (1995);J. Mellors et al, Science 272, 1167-1170 (1996).

Oligonucleotide probes, hybridization methodology, oxidizing agents andoxidation-reduction reaction methods, detection of oxidation reductionreactions, and apparatus useful for carrying out these methods are asgiven in sections A-H above.

J. Alternate Bases that are Redox Inactive

One disadvantage to the method described in section H above is that theoligonucleotide probe preferably does not contain a substantial numberof the preselected base (e.g., guanine). A solution to this problem isto use an alternate base that would substitute for guanine (i.e., a basethat, like guanine, has a greater binding affinity for cytosine than doother bases in a nucleic acid duplex) in the probe strand but would notbe oxidized by the oxidizing agent under the applicable reactionconditions. Examples of such alternate bases when guanine is thepreselected base are inosine and 7-deaza-guanine.

Thus, a method of detecting a target nucleic acid, where the targetnucleic acid contains at least one preselected base and the probe orcapture nucleic acid contains alternate redox inactive bases comprises:(a) contacting the target nucleic acid to a complementary nucleic acidthat specifically binds to the target nucleic acid to form a hybridizednucleic acid; (b) reacting the hybridized nucleic acid with a transitionmetal complex capable of oxidizing the preselected base in anoxidation-reduction reaction; (c) detecting the oxidation-reductionreaction; and (d) determining the presence or absence of the nucleicacid from the detected oxidation-reduction reaction at the preselectedbase. When the preselected base in the target nucleic acid is guanineand the target nucleic acid contains cytosine (which would ordinarilybond with guanine in the complementary nucleic acid), then thecomplementary nucleic acid contains an alternate base that bonds tocytosine in the hybridized nucleic acid. The alternate base may beselected from the group consisting of inosine and 7-deaza-guanine. Thereacting step typically comprises reacting the transition metal complexwith the nucleic acid under conditions sufficient to effect theselective oxidation of the preselected base without oxidizing thealternate base.

Oligonucleotide probes, hybridization methodology, oxidizing agents andoxidation-reduction reaction methods, detection of oxidation reductionreactions, and apparatus useful for carrying out these methods are asgiven in sections A-I above.

K. Polymerization of Preselected Base with Terminal Transferase

An alternative embodiment of the method described in section H aboveinvolves elongating the target nucleic acid with terminal transferase toprovide additional ones of the preselected base thereon. As illustratedin FIG. 7, such a method comprises: (a) contacting the test sample to anoligonucleotide probe that specifically binds to the target nucleic acidto form a hybridized nucleic acid, the oligonucleotide probe having endterminals that are blocked for elongation by terminal transferase; (b)contacting the oligonucleotide probe to a solution containing apreselected base in the presence of terminal transferase to produce anextension product of the target nucleic acid, with the extension productcomprised of the preselected base; (c) contacting the oligonucleotideprobe to a transition metal complex that oxidizes the preselected basein an oxidation- reduction reaction; (d) detecting the presence orabsence of the oxidation-reduction reaction; and (e) determining thepresence or absence of the target nucleic acid in the test sample fromthe detected oxidation-reduction reaction at the preselected base. Thetest sample is preferably separated from the oligonucleotide probe priorto the detecting step, and is more preferably separated from the probebetween steps (a) and (b) above. Separation may be carried out by use ofan immobilized probe, or the probe may be provided free in solution, asdiscussed in section H above.

Oligonucleotide probes, hybridization methodology, oxidizing agents andoxidation-reduction reaction methods, detection of oxidation reductionreactions, and apparatus useful for carrying out these methods are asgiven in sections A-I above.

L. Sandwich Assays

A further embodiment of the method of section H above is the so-called"sandwich" assay, schematically illustrated in FIG. 8. In a sandwichassay, the target nucleic acid is part of a three (or more) memberhybrid comprised of a capture probe, the target nucleic acid, and thesignal probe.

A method of detecting the presence or absence of a target nucleic acidin a test sample suspected of containing the same, comprises: (a)providing an oligonucleotide capture probe, wherein the capture probespecifically binds to the target nucleic acid; (b) contacting the testsample to the capture probe to form a hybridized nucleic acid; (c)contacting an oligonucleotide signal probe to the hybridized nucleicacid, wherein the signal probe specifically binds to the target nucleicacid therein, and wherein the signal probe contains at least onepreselected base, to produce a hybridized nucleic acid sandwich; (d)contacting the hybridized nucleic acid sandwich to a transition metalcomplex that oxidizes the preselected base in an oxidation-reductionreaction; (e) detecting the presence or absence of theoxidation-reduction reaction associated with the hybridized nucleicacid; and (f) determining the presence or absence of the target nucleicacid in the test sample from the detected oxidation-reduction reactionat the preselected base. The test sample is preferably separated fromthe capture probe, which separating step may occur between step (b) andstep (c) above, or between step (c) and step (d) above. Depending on theassay format (e.g., heterogenous or homogenous), the oligonucleotidecapture probe may be immobilized on a solid support (e.g., a polymericbead, a plate, or the inside surface of a microtiter plate well), oralternate means provided for separating the hybridized nucleic acid fromthe test sample, as discussed above.

Numerous "sandwich" assay formats are known. The choice of assay formatis not critical, and any suitable format may be employed to carry outthe present invention. For example, the oligonucleotide capture probemay be immobilized on a solid support, as described in U.S. Pat. No.4,486,539 to Ranki et al. The oligonucleotide probe may contain apolymer-forming unit, as described in U.S. Pat. No. 4,868,104 to Kurn etal., and the hybridized nucleic acid sandwich separated bypolymerization thereof. The signal probe may be linear or branched, asdescribed in M. S. Urdea, Clinical Chem. 39, 725-726 (1993). A mediatorpolynucleotide that binds the oligonucleotide capture probe to animmobilized polynucleotide, as described in U.S. Pat. No. 4,751,177 toStabinsky, may be employed. The oligonucleotide probe may be joined toone member of a specific binding pair (e.g., biotin), and the hybridizednucleic acid sandwich separated from the test sample by means of asecond binding interaction with the other member of the binding pair,that is immobilized on a solid support (e.g., avidin), as described inR. Goodson, EPO Application 0 238 332: W. Harrison, EPO Application 0139 489, and N. Dattagupta, EPO Application 0 192 168.

Oligonucleotide probes, hybridization methodotogy, oxidizing agents andoxidation-reduction reaction methods, detection of oxidation reductionreactions, and apparatus useful for carrying out these methods are asgiven in sections A-K above.

M. Detection of Preselected Base in the Presence of Background GuainineSignal

The presence of a preselected base in an oligonucleotide probe may bedetected even in the presence of background signal produced from theoxidation of guanine. Because the detection of mismatches relies uponthe ability to detect a preselected base in the oligonucleotide probe inthe presence of the the four native bases (A, T/U, C, and G). Therefore,the preselected base must be capable of being oxidized more rapidly thanthe other four bases.

The present invention provides an oligonucleotide probe useful for theelectrochemical detection of a preselected base in the presence ofbackground guanine signal. The oligonucleotide probe may consist of anyoligonucleotide probe as given in section B above, where at least onepurine base in the oligonucleotide probe is a purine substituent ofFormula I: ##STR1##

The oligonucleotide probe may contain as many bases of the foregoingformula as desired (e.g., 1, 2 or 3 up to 5, 10, or 15 or more)depending on the intended binding partner thereof. Specific examples ofsuch oligonucleotide probes, and nucleotides useful for the preparationthereof, are compounds of Formula II: ##STR2## wherein: R₁ isHO--P(O)(OH)--O--, a nucleotide, or an oligonucleotide;

R₂ is --H, a nucleotide or an oligonucleotide;

R₃ is --H, --OH, halo (e.g., fluoro, chloro), alkoxy (e.g., C1-C4 alkoxysuch as methoxy or ethoxy), amino, or azido; and

R₄ is --O-- or --CH₂ --.

Oligonucleotide probes as described in connection with Formulas I and IIabove are made in accordance with known techniques, modified in light ofthe Examples set forth below, as will he readily apparent to thoseskilled in the art.

In one preferred embodiment of the compound of Formula II, R₁ isHO--P(O)(OH)--O--. In another preferred embodiment of the compound ofFormula I, R is --H. When R₁ is a nucleotide or an oligonucleotide, thephosphodiester bond is to the 3' terminus. When R₂ is a nucleotide oroligonucleotide, the phospliodiesther bond is to the 5' terminus.

The compounds of Formula I are advantageously included as a base in anoligonucleotide probe which may be utilized in the methods of thepresent invention, as described in sections A-M above. Theoligonucleotide probe may of course include multiple bases, but shouldinclude at least one base of Formula I when the oligonucleotide probe isto be used for the detection of a preselected base in the presence ofbackground guanine. The oligonucleotide probe may be 5, 10, 50 or up to100 base pairs in length. A particular example of a compound of FormulaII is 6-mercaptoguanosine 5'-monophosphate (6-S-GMP).

N. Electrode Structures

An electrode useful for the electrochemical detection of a preselectedbase in a nucleic acid in accordance with the methods described abovecomprises: (a) a conductive substrate having a working surface formedthereon; and (b) a polymer layer connected to the working surface. Thepolymer layer is one that binds the nucleic acid (e.g., by hydrophobicinteraction or any other suitable binding technique) and is porous tothe transition metal complex (i.e., the transition metal complex canmigrate to the nucleic acid bound to the polymer). The conductivesubstrate may be a metallic substrate or a non-metallic substrate,including semiconductor substrates (e.g., gold, glassy carbon,indium-doped tin oxide, etc.). The conductive substrate may take anyphysical form, such as an elongate probe having a working surface formedon one end thereof, or a flat sheet having the working surface formed onone side thereof. The polymer layer may be connected to the workingsurface by any suitable means, such as by clamping the polymer layer tothe working surface, evaporation of a solution of the polymer onto theelectrode, or electropolymerizatiion. Exemplary polymers include, butare not limited to, nylon, nitrocellulose, polystyrene, andpoly(vinylpyridine). The thickness of the polymer layer is not critical,but can be from 100 Angstrom (Å) to 1, 10, or even 100 microns. Theelectrode may be used in essentially all of the methods described insections A-M above. Thus, in general, the present invention provides amethod of detecting a nucleic acid, said nucleic acid containing atleast one preselected base, the method comprising: (a) contacting asample containing said nucleic acid to an electrode, the electrodecomprising a conductive substrate having a working surface formedthereon and a polymer layer as described above connected to the workingsurface; (b) reacting the nucleic acid with a transition metal complexcapable of oxidizing the preselected base in an oxidation-reductionreaction; (c) detecting said oxidation-reduction reaction by measuringcurrent flow through said electrode; and (d) determining the presence orabsence of the nucleic acid from the detected oxidation-reductionreaction at the preselected base.

O. Microelectronic Devices

An advantage of the techniques described above is that they may becarried out with a microelectronic device. A microelectronic deviceuseful for the electrochemical detection of a nucleic acid species inthe methods described above comprises a microelectronic substrate havingfirst and second opposing faces; a conductive electrode on the firstface; and an oligonucleotide capture probe immobilized on the first faceadjacent the conductive electrode. The capture probe is spacedsufficiently close to the adjacent electrode (e.g., from about 0.1, 1,or 2μ up to about 50, 100, 500 or even 1000μ) so that an oxidationreduction reaction occuring at that probe, or at a target nucleic acidhybridized to that probe, is detected at the adjacent electrode.

In the preferred embodiment illustrated in FIG. 9 and FIG. 10, amicroelectronic device 20 has a plurality of separate electrodes 21 onthe first opposing face, and a plurality of separate oligonucleotidecapture probes 22 immobilized adjacent to each of the separateelectrodes. By providing a plurality of separate oligonucleotide probes,differing from one another, each with an associated electrode, a single,compact device is provided that can detect a variety of differenthybridization events. Each electrode is electrically connected to asuitable contact 23 so that the device may be wired or otherwiseoperatively associated with the necessary electronic equipment forcarrying out the detection and determining steps of the methodsdescribed herein.

The nucleic acid may be selectively immobilized at the appropriatelocation on the microelectronic substrate by known techniques. See,e.g., U.S. Pat. No. 5,405,783 to Pirrung et al. The microelectronicsubstrate may be a semiconductor (e.g., silicon) or non-semiconductormaterials that can be processed using conventional microelectronictechniques (e.g., glass). The electrode may be metal or a non-metallicconductive material, such as polycrystalline silicon. The electrode canbe formed using conventional microelectronic processing techniques, suchas deposition etching. A variety of suitable microelectronic structuresand fabrication techniques are well known to those skilled in the art.See, e.g., S. M. Sze, VLSI Technology (1983); S. K. Ghandhi, VLSIFabrication Principles (1983).

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In these examples, cm²/s means centimeters squared per second, M means molar concentration,M⁻¹ s⁻¹ means per molar per second, eV means electron volts, V meansvolts, nm means nanometers, GMP means guanosine 5'-monophosphate, andITO means tin-doped indium oxide electrode. Cyclic voltammograms werecollected with an EG+G Princeton Applied ResearchPotentiostat/Galvanostat, Model 273A, in accordance with knowntechniques. ITO working electrodes are fabricated from an ITO-coatedsoda-lime glass sheet, part number CH-50IN-1514, available from DeltaTechnologies, Ltd, 13960 North 47th Street, Stillwater, Minn. 55082-1234USA. Nylon film is available as HYBOND-N+ nylon membrane, catalog no.RPN 1210B, from Amersham Corp, 2636 Clearbrook Drive, Arlington Heights,Ill. 60005 USA.

EXAMPLE 1 Measurement of Cyclic Voltammogram of Ru(bpy)₃ ²⁺

The cyclic voltammograms of Ru(bpy)₃ ²⁺ with and without calf thymus DNAare shown in FIG. 1, with the catalytic enhancement produced by themultiple turnovers of oxidation of DNA by the oxidized form of the metalcomplex which are observed during a single voltammetric sweep. Thevoltammetry of any DNA-bound redox couple must be analyzed in terms of asquare-scheme that relates the bound and unbound forms because thediffusion coefficient of DNA is much lower (i.e., 2.0×10⁻⁷ cm² /s) thanthat of the metal complex (8.0×10⁻⁶ cm² /s). This phenomenon generallyleads to dramatically decreased currents for the bound form; however, atsufficient high ionic strength ([Na+]=0.8M), binding of the metalcomplex is too weak to influence the current response. In this case, thecurrent can be analyzed in terms of a simple EC' mechanism.

    Ru(bPy).sub.3.sup.2+ →Ru(bpy).sub.3.sup.3+ (E)

    Ru(bpy).sub.3.sup.3+ +DNA→Ru(bpy).sub.3.sup.2+ +DNA.sub.ox (C')

EXAMPLE 2 Analysis of Cyclic Voltammograms

Cyclic voltammograms were analyzed by fitting the completecurrent-potential curves, with the background subtracted, using theDIGISIM™ data analysis package. The input parameters were E_(1/2) forthe metal complex and the diffusion coefficients for the metal complexand the DNA, all of which were determined in separate experiments.Therefore, the sole parameter obtained from the fit was the second-orderrate constant for equation 2, k=9.0×10³ M³¹ 1 s⁻¹. This same rateconstant was determined over a wide range of scan rates.

The rate constant for oxidation of DNA by Ru(bpy)₃ ³⁺ was confirmed intwo separate experiments. First, square-wave voltammograms were used toobtain a pseudo-first-order k_(nbs) for equation 2 by fitting with theCOOL™ algorithm. The COOL™ algorithm uses a fitting approach that issignificantly different from DIGISIM™; nevertheless, plots of k_(nbs)against DNA were linear and gave a second-order rate constant k=8.2×10³M⁻¹ s⁻¹, which is in agreement with the rate constant obtained fromfitting cyclic voltammograms with DIGISIM™. Second, authentic samples ofRu(bpy)₃ ³⁺ were prepared and reacted with DNA directly in arapid-scanning stopped flow. Global analysis of the time-dependentspectra between 350 and 600 nm showed that Ru(bpy)₃ ³⁺ was convertedcleanly to Ru(bpy)₃ ²⁺ with no intermediates and a rate constant of12×10³ M⁻¹ s⁻¹. Thus, the rate constant for DNA oxidation by Ru(bpy)₃ ³⁺was firmly established by two independent electrochemical measurementswith dramatically different fitting protocols and by anon-electrochemical stopped flow technique with fitting of the completevisible spectra.

EXAMPLE 3 Analysis of Cyclic Voltammograms

If the driving force for electron transfer is significantly less thanthe reorganizational energy (λ), a plot of RT In k versus driving force(when corrected for work terms associated with approach of thereactants) should yield a straight line with a slope of 1/2. The rateconstants for oxidation of DNA by a number of Metal(bpy)₃ ³⁺ derivativeswith different redox potentials are shown in Table 1 below.

Since Marcus theory describes the driving-force dependence of theelectron-transfer rate, absolute rate constants can be analyzed in termsof the following equation:

    k=v exp[-β(r-r.sub.0)]exp[-(ΔG+λ).sup.2 /4λRT]

where v is the rate constant in the diffusion-controlled limit (10¹¹ M⁻¹s⁻¹), r is the distance between reactant and product in the activatedcomplex, r₀ is the distance of closest approach of reactant and product,and β describes the influence of the intervening medium. Incorporationof the guanine donor into the interior of the double helix imposes afinite distance across which the electron must tunnel to the dockedmetal complex, i.e., r≢r₀. However, if guanosine 5'-monophosphate (GMP)is used as the electron donor, direct collision of guanine with themetal complex is possible (r=r₀). For Fe(bpy)₃ ³⁺ and GMP, the rateconstant measured by stopped-flow is 2.6×10³ M⁻¹ s⁻¹. Known values of λfor related reactions are in the range 1-1.5 eV, which give a ΔG for theguanine^(+/0) couple of 1.1±0.1 V.

                  TABLE 1                                                         ______________________________________                                        Rate Constants for Oxidation of Guanine in DNA Oligomers                       by Ru(bpy).sub.3.sup.2+                                                        k(M.sup.-1 s.sup.-1).sup.a                                                              oligomer sequence       Δr.sub.Ru-6 (Å).sup.b           ______________________________________                                        1.2 × 10.sup.3                                                                  (5'-AAATATAGTATAAAA) ·                                                                       1.7 Å                                        (3'TTTATATCATATTTT) GC pair                                                  5.1 × 10.sup.3 (5'-AAATATAGTATAAAA) · 1.2 Å                 (3'-TTTATATTATATTTT) GT mismatch                                             1.0 × 10.sup.4c (5'-AAATATAGTATAAAA) · 1.0 Å                                               (3'-TTTATATGATATTTT) GG mismatch                                             1.9 × 10.sup.4 (5'-AAATATAGTATAAAA)                                     · 0.7 Å                            (3'-TTTATATAATATTTT) GA mismatch                                             1.8 × 10.sup.5 (5'-AAATATAGTATAAAA) single strand 0 Å                                              5.1 × 10.sup.3 (5'-AAATATAGTATAAAA                                     · 1.2 Å                             (3'-TTTATATCTCATTTT)                                                       ______________________________________                                         .sup.a DNA concentrations used to determine rate constants were based on      the moles of guanine nucleotides.                                             .sup.b Estimated distance of tunneling through solvent. Distances             calculated according to k/k.sub.ss = exp[-βΔr], where              β(H.sub.2 O) =3Å .sup.1 and k.sub.ss = 1.8 × 10.sup.5          M.sup.-1 s.sup.-1.                                                            .sup.c Since the rate constants are relative to guanine concentrations,       the observed rate for the GG mismatch has been normalized relative to the     other oligomers containing a single guanine.                             

In FIG. 2 are the cyclic voltammograms of Ru(bpy)₃ ²⁺ in the presence of5'-AAATATAGTATAAAA as a single stand (C) and hybridized to itscomplementary strand (A). As with GMP, r=r₀ for the single strand, andthe rate constant of 1.8×10⁵ M⁻¹ s⁻¹ gives ΔG(guanine^(+/0))=1.1 V andλ=1.3 eV, which are in agreement with the values from GMP oxidation.While there is a dramatic enhancement for the single strand, only aslight enhancement is observed for the fully hybridized duplex at thisscan rate, resulting in a four-fold reduction in current uponhybridization. Metal complexes such as Ru(bpy)₃ ²⁺ are known to dock toDNA in the minor groove, so the 150-fold slower rate constant (1.2×10³M⁻¹ s⁻¹) for oxidation of the duplex must result from the distancebetween the guanine residue and the surface-bound complex. When themetal complex is docked in the minor groove, guanine and the metalcomplex cannot come into intimate contact, and the electron must tunnelthrough the solvent that separates the guanine residue and the metalcomplex. Tunneling through water is much less efficient than throughnon-polar media, and the value of β for water is estimated to be about 3Å⁻¹. The tunneling distance can therefore be calculated according to:

    k/k.sub.ss =exp(-βΔr)

where Δr is the change in distance in the duplex compared to the singlestrand. From this analysis, Δr for the fully hybridized duplex is 1.7 Å.

The large value of β for water suggests that significant changes in theelectron-transfer rate constants will be effected by very small changesin the tunneling distance, which could in turn reflect smallperturbations in the DNA structure. Also shown in FIG. 2 is thevoltammogram of Ru(bpy)₃ ²⁺ in the presence of the same duplex where theGC base pair has been replaced by a GA mismatch. Incorporation of the GAmismatch results in a two-fold enhancement in the raw current comparedto the authentic duplex, which translates to a 16-fold change in rateconstant (k_(GA) =1.9×10⁴ M⁻¹ s⁻¹). The rate data for the single strand,fully hybridized duplex, and all three GX mismatches are set out inTable 1. Also shown are the calculated tunneling distances Δr relativeto the single strand. As expected, the guanine residue in G-purinemismatches is more accessible to the metal complex than in the GTmismatch where the two bases are still joined by two hydrogen bonds in awobble pair. Nonetheless, the GT mismatch still causes a 4-fold changein the rate constant, which is readily detectible. Therefore, theoxidation rate constants follow the trend G (single strand)>GA>GG>GT>GC.The ability to distinguish each of these mismatches from one another,provides the basis for mismatch-sensitive detection of hybridizationwhich is sensitive even to single-base pair mismatches at the base pairadjacent to the preselected base.

EXAMPLE 4 Modified Bases to Avoid Oxidation in Probe Strand:Substitution of Inosine for Guanine

Cyclic voltammograms were collected using an indium tin oxide (ITO)working electrode (area=0.32 cm²), Pt-wire counter electrode, and anAg/AgCl reference electrode. In FIG. 5, a sample containing 25 μMRu(bpy)₃ ²⁺ and 75 μM oligonucleotide dissolved in 50 mM Na-phosphatebuffer (pH 7) with 0.70M NaCl was scanned at 25 mV/s. In FIG. 6, asample containing 50 μM Ru(bpy)₃ ²⁺ and 0.3 mM of either 5'-GMP or5'-IMP dissolved in buffered aqueous solutions containing 700 mM NaCland 50 mM Na-phosphate buffer (pH=6.8, [Na⁺ ]=780 mM) was scanned at 2.5mV/s from 0.0 V to 1.3 V. Scans of mononucleotides in the absence ofRu(bpy)₃ ²⁺ showed no appreciable oxidative current. A freshly-cleanedITO electrode was used for each experiment, and a background scan ofbuffer alone subtracted from subsequent scans. Second-order guanineoxidation rate constants were determined by fitting of cyclicvoltammetric data to a two-step mechanism using the DIGISIM™ softwarepackage. All parameters other than the oxidation rate were determinedfrom a voltammograms of the metal complex alone on the same electrode.The 5'-GMP was purchased from Sigma and the 5'-IMP was purchased fromU.S. Biochemical, and both were used without further purification.Oligonucleotides were prepared in the UNC Department of Pathology andpassed through a 3000-molecular weight cutoff filter to removemononucleotides. Purity was assessed by reverse-phase HPLC. Theconcentration was determined from the optical absorption at 260 nm asdescribed in Fasman, G. D. CRC Handbook of Biochemistry and MolecularBiology; CRC Press, Boca Raton, Fla., 1975; Vol. 1. The hybrid in FIG. 5was prepared by heating the complementary strands of 90° C. for 5 minand slowly cooling to 25° C. over 2 h.

These data indicate that inosine may be substituted for guanine in theprobe strand to provide a redox inactive probe strand.

EXAMPLE 5 Modified Bases to Avoid Oxidation in Probe Strand:7-Deaza-Guanine

This example is carried out in essentially the same manner as example 4above, except that 7-deaza-guanine is used as the modified base as analternative to guanine to provide a redox-inactive probe strand.

7-deaza-guanine is oxidized at a rate of only 10³ M⁻¹ s⁻¹, which is twoorders of magnitude slower than guanine and is sufficiently slow toprovide a redox-inactive probe strand.

EXAMPLE 6 Detection Using Calf Thymus DNA Bound to Nylon MembraneAttached to ITO Electrode

Nylon film is cut into a circular shape, approximately 6 mm in diameterso as to fit into the electrochemical cell and cover the portion of theITO electrode exposed to the solution.

For the experiments in which only the cyclic voltammogram of the metalcomplex is obtained, the ITO electrode is first conditioned with buffer.The nylon disk (no DNA) is then inserted into the electrochemical celland 200 μL of a 200 μM metal complex solution is pipetted into the cell.For the Os(bpy)₃ ²⁺ experiments, an equilibration time of 6 minutes isused prior to electrochemical analysis. For the Ru(bpy)₃ ²⁺ experiments,an equilibration time of 15 minutes is used prior to electrochemicalanalysis. Cyclic voltammograms are collected using an PAR 273Apotentiostat at a scan rate of 25 mV/s.

For the DNA experiments, the DNA-soaked nylon disk is inserted into theelectrochemical cell after conditioning of the ITO electrode in theappropriate buffer. 200 μL of a 200 μM metal complex solution in theappropriate buffer is pipetted into the cell, and a cyclic voltammogramis taken after the appropriate equilibration time (6 minutes forOs(bpy)₃ ²⁺ and 15 minutes for Ru(bpy)₃ ²⁺) at a scan rate of 25 mV/s.The nylon disks are soaked for approximately 5 minutes in a solution of5.8 mM calf thymus DNA dissolved in water. A variety of soak times wereinvestigated ranging from 5 minutes to 18 hours. The DNA rapidly (withinminutes) associates with the nylon film, so short soak times aretypically employed. Under low salt conditions, a 50 mM Na-phosphatebuffer (pH=6.8,[Na⁺ ]=80 mM) is used. Under high salt conditions, a 50mM Na-phosphate buffer and 700 mM NaCl (pH=6.8,[Na⁺ ]=780 mM) solutionis used.

The cyclic voltammogram of Ru(bpy)₃ ²⁺ at the nylon-ITO electrode isshown in FIG. 11. The dashed line shows the voltammogram when the nylonmembrane is soaked in calf thymus DNA prior to attachment to theelectrode. There is a large catalytic current for the DNA-labelledmembrane that parallels that observed in solution. The experimentdemonstrates that Ru(bpy)₃ ²⁺ diffuses freely in the nylon film and thatdiffusion of the DNA is not required to realize the catalytic current.FIG. 11 also shows that a greater catalytic current is observed at lowersalt concentrations, due to enhanced interaction between the mediatorand the immobilized DNA.

FIG. 12A shows the same experiment using Os(bpy)₃ ²⁺ as the mediator.The osmium complex does not oxidize guanine, so any current enhancementobserved in the presence of guanine would have to arise frompreconcentration of the mediator due to DNA binding. In fact, thecurrent for Os(bpy)₃ ²⁺ is lower in the presence of DNA at the nylonelectrode than in the absence of DNA. The experiment demonstrates thatthe increased current for Ru(bpy)₃ ²⁺ when DNA is bound to the nylonelectrode is solely due to the proprietary catalytic reaction and notdue to a trival binding difference. The effect of salt concentration isshown in FIG. 12B, and is observed to be small compared to the largesalt effect observed for the catalytic reaction.

By binding the DNA to the nylon membrane attached to the ITO electrode,we have demonstrated that DNA may be detected even in the embodimentwherein the DNA is not diffusing hut the mediator is. This findingpermits the detection of DNA where the immobilized probes aresufficiently close to the electrode so that the probe-target hybridsreside in the diffusion layer of the mediator.

EXAMPLE 7 Detection of RNA Bound to Nylon Membrane Attached to ITOElectrode

The experiment is carried out as described in Example 6, except thattRNA from Bakers Yeast (purchased from Sigma) is used instead of calfthymus DNA. A nylon film disk was soaked in a solution of tRNA asdescribed in Example 6.

The cyclic voltammetry in the presence of Ru(bpy)₃ ²⁺ is shown in FIG.13. As with DNA, catalytic current is observed for both buffers withmore current at low salt. The difference observed in current between thehigh and low salt concentrations is not as dramatic as that observedwith DNA in Example 6 because tRNA does not bind cations as well as DNAand therefore the salt effects are less dramatic.

The results in FIG. 13 demonstrate that RNA can be detected in a manneridentical to that for DNA, which occurs because both RNA and DNA containguanine. The chemical composition of the sugar-phosphate backbonetherefore does not influence the catalytic current. Based upon thisobservation, the detection of both single- and double-stranded DNA andRNA, DNA-RNA hybrids, as well as single strands or duplexes containingother modified backbones such as PNA's, carbocycles, phosphorothioates,or other substituted ribose linkages is possible.

EXAMPLE 8 Detection of RNA

For quantitative detection of RNA, a DNA (or RNA, PNA, or otheralternative backbone) probe is immobilized on a solid support. The probemay be modified to be redox-inert by substitution of inosine or7-deaza-guanine for the guanines in the probe strand. The immobilizedprobe is then contacted with a solution of target RNA (for example fromHIV or Hepatitis C). The solid surface then contains a redox-inert,immobilized probe hybridized to a strand of RNA. The solid surface isthen contacted with a solution of Ru(bpy)₃ ²⁺, and the cyclicvoltammogram of the mediator is measured. The catalytic current signalsthe hybridization event, and the magnitude of the current is used toquantitate the bound RNA strand based upon the known number of guaninesin the strand.

For the mismatch detection of RNA, a DNA (or RNA, PNA, or otheralternative) probe is immobilized to a solid surface. The preselectedbase in the probe strand is oxidized more easily than the other bases.The surface is contacted with a solution of target RNA and thencontacted with a solution of Ru(bpy)₃ ²⁺ or other mediator. The extentof hybridization (perfect match, no pairing, or mismatch) is thendetermined at the preselected base in the same manner as for DNA.

EXAMPLE 9 Detection of a Preselected Sequence of Bases

The method was carried out as described in Example 3. The cyclicvoltammograms set forth in FIG. 14 demonstrate that the current due to5'-G is much less than that for 5'-GG which is much less than that for5'-GGG. This tremendous increase in current is observed for both singlestrands and duplexes that contain GG and GGG sequences. The increase incurrent is not due simply to the increase in the number of G's, becauseas shown in FIG. 15, the increase in current due to adding G's to thesame strand is much lower if the G's are interspersed. Since the 5'-G ofthe GGG is much easier to oxidize than a single G, it is possible toselect a mediator (with a lower redox potential) that is capable ofoxidizing GGG but not G.

The cyclic voltammogram of Ru(4,4'-dimethyl-bipyridine)₃ ²⁺ is shown inFIG. 16 along with repeat scans in the presence of the single Goligonucleotide and the GGG oligonucleotide. As shown, catalytic currentis observed only in the presence of the GGG oligonucleotide. Thisexample shows the ability to tune the potential of the mediator suchthat a more easily oxidized sequence can be detected in the presence ofbackground guanine. The same strategy can be applied to detecting asingle synthetic base that is derivatized to make it more easilyoxidized than guanine.

The experiment demonstrates that it is possible to lower the potentialof the mediator and still distinguish a more easily oxidizable base orbase sequence.

EXAMPLE 10 Detection of a Preselected Guanine Derivative in the Presenceof Background Native Guanine

The disodium salt of 6-mercaptoguanosine 5'-monophosphate (6-S-GMP)##STR3## is prepared by phosphorylation of commercially available6-mercaptoguanosine (from Sigma). The phosphorylation is performed usingPOCl₃ according to the procedure of M. Yoshikawa, et al., Bull. Chem.Soc. Jpn. 42:3505 (1969). The disodium salt of 6-S-GMP is purified byHPLC prior to voltammetric analysis. Cyclic voltammograms are performedat high ionic strength as in the inosine-5'-monophosphate example. Theworking electrode is a ITO with a Hybond N+ nylon membrane attached tothe surface to prohibit direct oxidation of the 6-S-GMP. The counterelectrode is a Pt wire. The reference electrode is Ag/AgCl. The scanrate is 25 mV/s.

The results of the cyclic voltammogram are set forth graphically in FIG.17, where curve A shows Ru(4,4'-Me₂ -bpy)₃ ²⁺ alone (4,4'-Me₂-bpy=4,4-'dimethyl-2,2'-bipyridine). Upon addition of 5'-GMP, noenhancement of the Ru(Me₂ bpy)₃ ²⁺ wave is observed; however, additionof 6-mercaptoguanosine 5'-monophosphate (6-S-GMP) leads to a dramaticcurrent enhancement (curve B). The peak current in the presence of5'-GMP is identical to that in curve A. The data demonstrate that it ispossible to detect 6-mercaptoguanine bases in the presence of backgroundnative guanine.

EXAMPLE 11 Detection of DNA Hybridization with the Preselected Base onthe Target Strand

Nylon membranes (Hybond N+, Amersham, 480-600 μg/cm²) are cut intocircular shapes, approximately 6 mm in diameter. The nylon disks areplaced in a concentrated solution of polycytidylic acid (available fromSigma) in water and allowed to soak for 1 hour. The disks are thenremoved from the polycytidylic acid (poly[C]) solution and placed onparafilm and allowed to dry. As the disks are drying, an additional 15μL of poly[C] solution is added to the films in three 5 μL aliquots. Thedisks are allowed to dry completely. The dried nylon disks are thenwashed in the low salt buffer (50 mM Na-phosphate, pH=6.8, [Na⁺ ]=80 mM)to remove any poly[C] which is not strongly bound during the soakingprocess.

As a control experiment, a poly[C]-impregnated disk is put through amock hybridization procedure in which it is not exposed to anyadditional nucleic acid, but is exposed to all other hybridizationsteps. The disk is placed in 400 μL of milli-Q water, heated at 48° C.for 1 hour, and allowed to cool to room temperature. The disk is removedfrom the water and washed in low salt buffer prior to electrochemicalanalysis. Disks prepared in this manner represent the background scans(A) in both FIGS. 18 and 19.

A poly[C]-impregnated disk is placed in 400 μL of a polyguanylic acid(available from Sigma) in water solution, heated at 48° C. for 1 hour,and allowed to cool to room temperature. The disk is then removed fromthe polyguanylic acid (poly[G]) solution and washed in a low salt bufferprior to electrochemical analysis.

Calf thymus DNA (available from Sigma) in water is denatured (melted) byheating to 90° C. for 10 minutes. A poly[C]-impregnated disk is placedin the denatured calf thymus DNA solution, heated to 48° C. for 1 hour,and allowed to cool to room temperature. The disk is removed from thecalf thymus DNA solution and washed in low salt buffer prior toelectrochemical analysis. As a control, a nylon disk which has not beenimpregnated with poly[C] is also subjected to the same procedure.Binding and detection of calf thymus DNA by adsorption into the nylonfilm (not by hybridization) is observed in the control membrane.

The nylon disk, treated as described above, is inserted into theelectrochemical cell, after the conditioning of the ITO electrode withthe low salt buffer. 200 μL of a 200 μM Ru(bpy)₃ ²⁺ solution is pipettedinto the cell and a cyclic voltammogram is taken after a 15 minuteequlibration time. The scan rate is 25 mV/sec.

The cyclic voltammogram is reported in FIG. 18. The probe sequencepoly[C] is immobilized onto a Hybond N+ nylon membrane and thehybridization protocol is carried out in buffer ("mock hybridization").The membrane is attached to the ITO working electrode and a cyclicvoltammogram of Ru(bpy)₃ ²⁺ is obtained (A). The membrane is immersed ina solution of poly[G] and hybridization is performed according to thesame protocol. The cyclic voltammogram ,of Ru(bpy)₃ ²⁺ is then measured(B), and a large current enhancement is obtained due to catalyticoxidation of the hybridized poly[G] target. As shown in FIG. 19, theassay is specific for the appropriate sequence. FIG. 19 compares thevoltammetry for the poly[C] membrane where the hybridization procedurewas carried out in buffer (A) or in a solution of single-stranded calfthymus DNA (B). FIG. 19 shows that if the target sequence is notpresent, no current enhancement is obtained.

EXAMPLE 12 Detection of DNA at Nylon-Modified Glassy Carbon Electrodes

FIG. 20 shows the cyclic voltammogram (or "CV") of a glassy carbonelectrode with a nylon film attached before (A) and after (B)immobilization of DNA on the nylon film.

Nylon membrane (Zeta-Probe, Bio-Rad, 80-100 μg/cm²) was cut intocircular shapes, approximately 5 mm in diameter. The nylon disk asfashioned covers the glassy carbon electrode surface and is held inposition by a plastic sleeve. For the experiments in which only the CVof the metal complex was obtained, the glassy carbon electrode was firstconditioned with a low salt, 50 mM Na-phosphate buffer (pH=6.8, [Na⁺]=80 mM). The nylon disk (no DNA) was then attached to the electrode and400 μL of a 200 μM Ru(bpy)₃ ²⁺ solution was pipetted into theelectrochemical cell. An equilibration time of 15 minutes was used priorto electrochemical analysis. Cyclic voltammograms were collected usingan PAR 273A potentiostat using a scan rate of 25 mV/s. In a typical DNAexperiment, the glassy carbon electrode is first conditioned in the lowsalt, Na-phosphate buffer. A nylon disk was soaked for approximately 5minutes in a solution of 5.8 mM calf thyinus DNA dissolved in water. Thedisk was then removed from the solution and positioned over the glassycarbon electrode using the sleeve to hold it in place. 400 μL of a 200μM (Ru(bpy)₃ ²⁺ was pipetted into the electrochemical cell and after a15 minute equilibration a cyclic voltammogram was taken using a scanrate of 25 mV/s.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 11                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -      (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Johnston, - #D. H.                                                    Glasgow, - #K. C.                                                             Thorpe, H - #. H.                                                        (B) TITLE: Electrochemical - # Measurement of the Solvent                         Accessibilit - #y of Nucleobases Using Electron                               Transfer - #between DNA and Metal Complexes                              (C) JOURNAL: J. Am. - #Chem. Soc.                                             (D) VOLUME: 117                                                               (E) ISSUE: 35                                                                 (F) PAGES: 8933-8938                                                          (G) DATE: 1995                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - AAATATAGTA TAAAA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #binding                                          (B) LOCATION: complement - #(1..15)                                           (D) OTHER INFORMATION: - #/function= "correctly matched at                         the G - #of bound moiety"                                                     /bound.sub.-- - #moiety= "AAATATAGTATAAAA"                      - -      (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Johnston, - #D. H.                                                    Glasgow, - #K. C.                                                             Thorp, H. - # H.                                                         (B) TITLE: Electrochemical - # Measurement of the Solvent                          Accessibilit - #y of Nucleobases Using Electron                               Transfer - #between DNA and Metal Complexes                              (C) JOURNAL: J. Am. - #Chem. Soc.                                             (D) VOLUME: 117                                                               (E) ISSUE: 35                                                                 (F) PAGES: 8933-8938                                                          (G) DATE: 1995                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TTTTATACTA TATTT              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #binding                                          (B) LOCATION: complement - #(1..15)                                           (D) OTHER INFORMATION: - #/function= "GA mismatch at base 8                        of bound - #moiety"                                                           /bound.sub.-- - #moiety= "AAATATAGTATAAAA"                      - -      (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Johnston, - #D. H.                                                    Glasgow, - #K. C.                                                             Thorp, H. - # H.                                                         (B) TITLE: Electrochemical - # Measurement of the Solvent                          Accessibilit - #y of Nucleobases Using Electron                               Transfer - #between DNA and Metal Complexes                              (C) JOURNAL: J. Am. - #Chem. Soc.                                             (D) VOLUME: 117                                                               (E) ISSUE: 35                                                                 (F) PAGES: 8933-8938                                                          (G) DATE: 1995                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TTTTATAATA TATTT              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -      (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Napier, M. - # E.                                                     Loomis, C - #. R.                                                             Sistare, - #M. F.                                                             Kim, J.                                                                       Eckhardt, - #A. E.                                                            Thorp, H. - # H.                                                         (B) TITLE: Probing Biom - #olecule Recognition with Electron                       Transfer: - #Electrochemical Sensors for DNA                                  Hybridizatio - #n                                                        (C) JOURNAL: Bioconjugate - #Chemistry                                        (D) VOLUME: 8                                                                 (E) ISSUE: 6                                                                  (F) PAGES: 906-913                                                            (G) DATE: 1997                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GGGAAATATA GTATAAAAGG G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - AAATATAGGT ATAAAA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - AAATATAGGG TATAAAA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AAATATAGTA GTATAAAA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - AAATATAGTA GTAGTATAAA A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #binding                                          (B) LOCATION: complement - #(1..15)                                           (D) OTHER INFORMATION: - #/function= "GT mismatch at base 8                        of bound - #moiety"                                                           /bound.sub.-- - #moiety= "AAATATAGTATAAAA"                      - -      (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Johnston, - #D. H.                                                    Glasgow, - #K. C.                                                             Thorp, H. - # H.                                                         (B) TITLE: Electrochemical - # Measurement of the Solvent                          Accessibilit - #y of Nucleobases Using Electron                               Transfer - #between DNA and Metal Complexes                              (C) JOURNAL: J. Am. - #Chem. Soc.                                             (D) VOLUME: 117                                                               (E) ISSUE: 35                                                                 (F) PAGES: 8933-8938                                                          (G) DATE: 1995                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - TTTTATATTA TATTT              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #binding                                          (B) LOCATION: complement - #(1..15)                                           (D) OTHER INFORMATION: - #/function= "GG mismatch at base 8                        of bound - #moiety"                                                           /bound.sub.-- - #moiety= "AAATATAGTATAAAA"                      - -      (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Johnston, - #D. H.                                                    Glasgow, - #K. C.                                                             Thorp, H. - # H.                                                         (B) TITLE: Electrochemical - # Measurement of the Solvent                          Accessibilit - #y of Nucleobases Using Electron                               Transfer - #between DNA and Metal Complexes                              (C) JOURNAL: J. Am. - #Chem. Soc.                                             (D) VOLUME: 117                                                               (E) ISSUE: 35                                                                 (F) PAGES: 8933-8938                                                          (G) DATE: 1995                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - TTTTATAGTA TATTT              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: YES                                                - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #binding                                          (B) LOCATION: complement - #(1..15)                                           (D) OTHER INFORMATION: - #/function= "Base mismatch at bases                       8-9 of - #bound moiety"                                                       /bound.sub.-- - #moiety= "AAATATAGTATAAAA"                                    /citation=- # ([1])                                             - -      (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Johnston, - #D. H.                                                    Glasgow, - #K. C.                                                             Thorp, H. - # H.                                                         (B) TITLE: Electrochemical - # Measurement of the Solvent                          Accessibilit - #y of Nucleobases Using Electron                               Transfer - #between DNA and Metal Complexes                              (C) JOURNAL: J. Am. - #Chem. Soc.                                             (D) VOLUME: 117                                                               (E) ISSUE: 35                                                                 (F) PAGES: 8933-8938                                                          (G) DATE: 1995                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - TTTTATTCTA TATTT              - #                  - #                      - #    15                                                                 __________________________________________________________________________

What is claimed is:
 1. A microelectronic device useful for theelectrochemical detection of a member of a binding pair, said devicecomprising:a microelectronic substrate; a conductive oxidation-reductiondetection electrode on said substrate; and a first member of a specificbinding pair immobilized on a non-conductive layer and capable ofreacting with a second member of the specific binding pair present in asample, said first member being adjacent said detection electrode sothat an oxidation-reduction reaction occurring upon application of apotential to the detection electrode is detectable.
 2. Themicroelectronic device according to claim 1, wherein the microelectronicsubstrate comprises a sample container, which comprises said conductiveoxidation-reduction detection electrode and said immobilized firstmember.
 3. The microelectronic device according to claim 2, wherein themicroelectronic substrate comprises said sample container whichcomprises a plurality of conductive oxidation-reduction detectionelectrodes and a plurality of immobilized first members.
 4. Themicroelectronic device according to claim 3, further comprising aconductive reference electrode comprising a conducting metal.
 5. Themicroelectronic device according to claim 3 or 4, further comprising aconductive auxiliary electrode comprising a conducting metal.
 6. Themicroelectronic device according to claim 5, wherein theoxidation-reduction reaction is detectable via an electrical connectionfrom each conductive oxidation-reduction detection electrode.
 7. The (A)microelectronic device according to claim 6, wherein said substrate issilicon.
 8. The microelectronic device according to claim 6, wherein anymember within the plurality of first members of specific binding pairsmay be the same as or different from any other member within theplurality of first members.
 9. The microelectronic device according toclaim 8, wherein each member within the plurality of first members ofspecific binding pairs is different from every other member within theplurality of first members.
 10. The microelectronic device according toclaim 6, wherein said first member is selected from the group consistingof nucleic acids and proteins.
 11. The microelectronic device accordingto claim 6, further comprising an oxidation-reduction reaction detector.12. The microelectronic device according to claim 6, wherein thesubstrate is glass.
 13. The microelectronic device according to claim 6,wherein said microelectronic device comprises more than one samplecontainer.
 14. The microelectronic device according to claim 13, whereinthe plurality of first members of specific binding pairs within anysample container may be the same as or different from the plurality offirst members in any other sample container.
 15. The microelectronicdevice according to claim 14, wherein the plurality of first members ofspecific binding pairs in each sample container is the same as theplurality of first members in every other sample container.
 16. Themicroelectronic device according to claim 13, wherein any member withinthe plurality of first members within a particular sample container maybe the same as or different from any other member in said particularsample container.
 17. The microelectronic device according to claim 16,wherein each member within the plurality of first members within aparticular sample container is different from every other member in saidparticular sample container.
 18. The microelectronic device according toclaim 13, wherein each sample container comprises a microelectronicsubstrate, a conductive oxidation-reduction detection electrode and animmobilized first member of the specific binding pair.
 19. Themicroelectronic device according to claim 13, wherein said first memberis selected from the group consisting of nucleic acids and proteins. 20.The microelectronic device according to claim 19, wherein said firstmember of a specific binding pair comprises an oligonucleotide captureprobe, and said oligonucleotide capture probe is from 4 to 100nucleotides in length.
 21. The microelectronic device according to claim13, further comprising an oxidation-reduction reaction detector.
 22. Themicroelectronic device according to claim 13, wherein said substrate issilicon.
 23. The microelectronic device according to claim 13, whereinsaid substrate is glass.
 24. The microelectronic device according toclaim 6, wherein said first member of a specific binding pair comprisesan oligonucleotide capture probe, and said oligonucleotide capture probeis from 4 to 100 nucleotides in length.
 25. The microelectronic deviceaccording to claim 6:wherein each of the conductive oxidation-reductiondetection electrodes is electrically connected to a potentiost; andwherein the sample container comprises the sample and a solution of anelectrolyte, such that each of the detection electrodes is inelectrochemical contact with the solution, which contains anelectrochemical mediator capable of participating in anoxidation-reduction reaction with the electrodes under conditionswhereby an electrical potential is applied to the electrodes.
 26. Themicroelectronic device according to claim 25, further comprising anoxidation-reduction reaction detector for detecting saidoxidation-reduction reaction, wherein when the sample contains thesecond member of the specific binding pair, more current is detectablethan when the sample does not contain the second member of the specificbinding pair.
 27. The microelectronic device according to claim 25,wherein the microelectronic device comprises a plurality of samplecontainers, each of said sample containers comprising a solution of anelectrolyte, having said conductive oxidation-reduction detectionelectrodes in electrochemical contact with the solution, and having anelectrochemical mediator in the solution.